Abstract My long-term goal is to understand intracellular signaling cascades and their contribution to image processing in retina. As the eye flicks about a scene, a photoreceptor sees an alternating pattern of light and dark. Correspondingly, the photoreceptor transiently decreases and increases its glutamate release. Each pulse of glutamate has two effects: in OFF bipolar cells, it directly opens an AMPA/kainate cation channel, and in ON bipolar cells, it activates the metabotropic receptor, mGluR6, that indirectly closes a cation channel. The light response signaled by the ON bipolar cell is crucial for night vision, and subserves half the dynamic range in day vision. Though central to retinal processing, the basic molecular mechanisms that underly the the light ON response are still enigmatic, and are therefore the focus of this proposal. We previously showed that the subunit of the heterotrimeric G-protein, Go1, is required for the ON response. However, which G and which G isoforms comprise the other two subunits of this G- protein is unknown. Once Go is activated, either of its activated arms, Go1GTP or the free G dimer, can lead to channel closure, but which one does so is yet unknown. The next step of the cascade was thought to involve cGMP as a second messenger, but recent evidence suggests cGMP is a modulator. Still, whether cGMP activates a kinase to phosphorylate the receptor or the channel is controversial. Here we propose three Aims to answer these questions. In Aim 1, based on our profiling of ON bipolar cells and published immunocytochemistry, we hypothesize that the G-protein mediating the light ON response is Go1313. Aim 1 will test this hypothesis by using RNA interference to silence the genes that encode G3 and G13. Specific shRNA vectors will be injected subretinally to postnatal P0-P2 mice and transfected to bipolar cells by electroporation. At P21-P40, the compound light response of ON bipolar cells will be recorded using electroretinograms, and a single rod bipolar cell's response will be recorded with whole cell configuration. Aim 2 will determine which arm of Go leads to channel closure by uncoupling Go1 from the G dimer. We will record from a rod bipolar cell and dialyze either G-activating peptides to activate G, G scavengers to deactivate G, or active Go1 to test its direct effect. During dialysis we will monitor the agents' effects on holding current, input resistance, and light response. In Aim 3, we will first study cGMP's effect on the mGluR6 cascade in oocytes expressing the appropriate proteins. We will then test the hypothesis that cGMP's synthetic enzyme is guanylyl cyclase 11 and hydrolyzing enzyme is phosphodiesterase type 9A. Once identified, we will use knockout mice to study how these genes' deletion affect the rod bipolar light responses. Our research will contribute to the fundamental knowledge of the first synapse on the visual pathway and will identify new molecular players whose mutations may lead to night blindness, thus extending the basic foundation for future clinical studies.